Erythrocyte ankyrin, Ank-1, is expressed in nonerythroid tissues including brain and skeletal muscle. Northern blot analysis using erythroid region-specific ANK-1 probes revealed that skeletal muscle transcripts are recognized only by probes from the 3′ end of the erythroid cDNA. 5′ RACE using an antisense primer in the 3′ untranslated region of the erythroid transcript and human skeletal muscle RNA as template identified a product of ≈850bp in length. Nucleotide sequence analysis of this transcript revealed a novel 5′ end, including an initiator methionine, followed by 370bp of 3′ sequence previously identified in erythroid transcripts. Deduced amino acid sequence of this transcript predicted a peptide 149AA in length with a hydrophobic NH2-terminus. Database searching did not reveal any significant homology of the novel 5′ end to known sequence. Northern blot analysis using the novel sequence as probe identified transcripts of 2.4 and 1.6 in skeletal and cardiac muscle. Clones obtained from a human skeletal muscle cDNA library revealed that alternate polyadenylation is the basis of these transcripts. Analysis of a human genomic DNA clone revealed that the coding sequences are composed of four exons spread over ≈10kb of DNA with complex alternate splicing of the penultimate exon. The 5′ flanking DNA contained a TATA box 27bp upstream of the putative cap site. To identify cis-acting sequences essential for transcription of this promoter, human tissue culture cell lines HeLa, K562 (erythroleukemia) and the murine tissue culture cell line C2C12 (myoblast) were transfected with plasmids containing the 5′ flanking sequences of the muscle transcript fused to a luciferase reporter gene. There was no expression in HeLa or K562 cells. In C2C12 cells, a 186bp fragment directed muscle-specific activity and contained consensus sequences for several potential DNA-binding proteins including two E boxes and an SP1 site. DS oligonucleotides containing each of these three potential binding sites produced a band on gel shift analysis using C2C12 nuclear protein extracts. MyoD transactivated the 186bp promoter fragment in a dose-dependent fashion in HeLa and K562 cells. These results may have implications for patients with coexistent hereditary spherocytosis and skeletal muscle abnormalities and/or cardiomyopathy.